Prior studies with halogenated pyrimidines such as the fluorinated pyrimidines 1-(2'-deoxy-2'-fluoro-1-.beta.-D-arabinofuranosyl)-5-iodouracil (FIAU) and 1-(2'-deoxy-2'-fluoro-1-.beta.-D-arabinofuranosyl)-5-methyl uracil (FMAU) have indicated that these compounds are highly effective inhibitors of simian varicella virus both in vitro and in vivo in African green monkeys. Soike et al., Antimicrob. Agents Chemother. 29: 20-25 (1986). FIAU at oral doses of 3 mg/kg/day prevented development of simian varicella rash in monkeys and appreciably reduced viremia while FMAU was highly effective in preventing rash and viremia in monkeys at doses as low as 0.2 mg/kg/day. Unfortunately central nervous system toxicity observed in patients with advanced cancer treated with FMAU restricted its further consideration for antiviral applications.
Synthesis of analogs of FMAU resulted in the selection of the ethyl analog 1-(2'-deoxy-2'-fluoro-1-.beta.-D-arabinofuranosyl)-5-ethyl uracil (FEAU) as a potential antiviral agent with a more favorable therapeutic index. Chou et al., Antimicrob. Agents Chemother. 31:1355-1358 (1987). Although FEAU was slightly less active than FMAU in inhibiting herpes simplex virus infection in Vero cells and in mice, FEAU was also much less toxic. Fox et al., in Herpes Viruses and Virus Chemotherapy, pp. 53-56 (Elsevier Science Publishing, Inc., New York, 1985).
It has been shown that FEAU, as with FIAU and FMAU, is a highly effective inhibitor of simian varicella virus in the African green monkey. Activity of FEAU against simian varicella virus was comparable to that reported for FIAU but less than FMAU.
Similar levels of activity have been reported for the fluorinated pyrimidines in the treatment of herpes simplex virus infection in other animal models, U.S. Pat. No. 4,666,892. Trousdale et al., Antimicrob. Agents Chemother. 23:808-813 (1983) showed topical treatment of rabbit eyes infected with the McKrae strain of HSV-1 with solutions of FMAU reduced corneal infection. Mice infected by intracerebral inoculation of HSV-1 (KOS strain) were protected from death by both 1-(2'-deoxy-2'-fluoro-1-.beta.-D-arabinofuranosyl)-5-iodocytosine (FIAC) and FMAU. Schinazi et al., Antimicrob. Agents Chemother. 24:95-103 (1983). FIAC and FMAU were also successful in treating HSV-2 infection in guinea pigs, although higher doses were required. Mayo et al., Antimicrob. Agents Chemother. 26:354-357 (1984). Evaluation of FEAU in these animal models has not been reported.
Halogenated pyrimidines such as the fluorinated pyrimidines are highly effective antiviral and would be expected to have great potential for the treatment of human viral infections. FEAU was of particular interest because of its reported low toxicity. It was therefore attempted to determine whether the effective antiviral dose of FEAU might be further reduced by combination of FEAU with another antiviral active against simian varicella virus.
Interferons constitute a group of naturally occurring proteins which possess antiviral, antitumor and immunoregulatory properties. Two types of interferons have been identified based upon differences in their observed biological properties and molecular structures: Type I and Type II. Of particular interest was interferon-beta, a Type I interferon which could be induced in fibroblasts by viral challenge and contained 165 amino acids. Because human native interferon is expensive to extract, techniques have been developed for preparing recombinant forms of human interferon. Sugano et al., European Patent Publication No. 28,033 published June 6, 1981, have developed a technique for inserting a recombinant plasmid containing the human interferon-beta gene into a host microorganism, preferably E. coli, to obtain a transformant which expresses recombinant interferon-beta. However, the transformed E. coli contains multimers so purification and isolation of the interferon-beta is difficult. European Patent Publication No. 109,748 published May 30, 1984 describes a human interferon-beta mutein (IFN.beta..sub.ser17) which has a cysteine residue at amino acid position 17 replaced by a serine residue to eliminate the problems of purification, along with improving the potency and stability of interferon-beta. Human IFN.beta..sub.ser17 has been combined with 9-(1,3-dihydroxy-2-propoxy)methyl guanine (DHPG) to form a composition synergistically effective in inhibiting herpes simplex virus (HSV-2) as reported by Eppstein et al. in Biochemical and Biophysical Research Communications 120: 66-73 (1984). More recently it was shown by Soike et al. in Journal of Infectious Diseases 156: 607-614 (1987) that combining low doses of human recombinant interferon-beta (Hu rIFN.beta.) with DHPG reduced the effective dose of DHPG tenfold.
In view of FEAU's low toxicity and effectiveness in treating viral infections and the established antiviral properties of Hu rIFN.beta., it would be useful to investigate whether their combination would provide a synergistic effect in treating human viral infections, using the African green monkey as an animal model.